Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB1)-receptors. The present study examined whether CB1-receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 microg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 microg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB1-receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB1-receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.